The effect of polylysine on casein‐kinase‐2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme

The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic α subunit, either alone or in combination with the recombinant non‐catalytic β subunit. Calmodulin is not phosphorylated by the CK2 holoenzyme, in either the native or the reconstituted form, unless polylysine is added. In the presence of polylysine, it becames a good substrate for CK2 (Km, 14.2 μM, Kcat 4.6 mol · min−1· mol CK2−1). The recombinant α subunit, however, spontaneously phosphorylates calmodulin, this phosphorylation being actually inhibited rather than stimulated by polylysine. The calmodulin tridecapeptide, RKMKDTDSEEEIR, reproducing the phosphorylation site for CK2, is spontaneously phosphorylated by either CK2 holoenzyme or the recombinant α subunit with 5.8‐fold and 2.8‐fold stimulation by polylysine, respectively. The recombinant β subunit of CK2 is itself a good exogenous substrate for the enzyme, its phosphorylation, however, is inhibited rather than enhanced by polylysine. On the contrary, the phosphorylation of the nonapeptide, MSSSEEVSW, reproducing the β‐subunit phosphoacceptor site, is dramatically stimulated by polylysine. Using a variety of small peptide substrates, it was shown that phosphorylation rate is diversely stimulated by polylysine. The observed stimulation, moreover, is variably accounted for by changes in Vm and/or Km depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the β subunit, since the recombinant a subunit is much less responsive than CK2 holoenzyme, either native or reconstituted. While the phosphorylation of the peptide RRRDDDSDDD by CK2 is stimulated 2.8‐fold, with 15 nM polylysine being required for half‐maximal stimulation, a stimulation of only 1 Sfold, with 80 nM polylysine required for half‐maximal stimulation, is attained with recombinant α subunit. The concentration of polylysine required for half‐maximal stimulation is comparable to CK2 concentration and increases by increasing CK2 concentration, suggesting that polylysine primarily interacts with the enzyme, rather than with the peptide substrate.